S. E. Moskalenko

Saint Petersburg State University, Sankt-Peterburg, St.-Petersburg, Russia

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Publications (16)16.48 Total impact

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    ABSTRACT: A search for proteins which interact with the eRF1 termination translation factor encoded by SUP45 gene in yeast Saccharomyces cerevisiae was carried out by screening yeast genes located on a multicopy plasmid. This approach allowed finding the ECM23 gene whose increased expression led to lower viability of the sup45 nonsense mutants. Overexpression ECM23 gene led also to an antisuppressor effect, although the amount of the eRF1protein was not changed. Possible mechanisms of how ECM23 can affect the viability of the sup45 nonsense mutants are discussed.
    03/2014; 4(2):131-136. DOI:10.1134/S207905971402004X
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    O. A. Murina, S. E. Moskalenko, G. A. Zhouravleva
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    ABSTRACT: At present, the machinery supporting the viability of organisms possessing nonsense mutations in essential genes is not entirely understood. Nonsense mutants of Saccharomyces cerevisiae yeast containing a premature translation termination codon in the essential SUP45 gene are known. These strains are viable in the absence of mutant suppressor tRNAs; hence, the existence of alternative mechanisms providing nonsense suppression and mutant viability is conjectured. Analysis of clones obtained by transformation of a strain bearing a nonsense-mutant allele of SUP45 with a multicopy yeast genomic library revealed three genes encoding wild-type tRNATyr and four genes encoding wild-type tRNAGln, which increased nonsense mutant viability. Moreover, overexpression of these genes leads to an increase in the amount of the full-length eRF1 protein in cells and compensates for heat sensitivity in the nonsense mutants. Probable ways of tRNATyr and tRNAGln influence on the increase in the viability of strains with nonsense mutations in SUP45 are discussed. Key wordstranslation termination-yeast-nonsense suppression- SUP45 - sup45 nonsense mutants-eRF1-tRNA
    Molecular Biology 04/2010; 44(2):268-276. DOI:10.1134/S0026893310020123 · 0.74 Impact Factor
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    O. A. Murina, S. E. Moskalenko, G. A. Zhouravleva
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    ABSTRACT: 301 *Ключевыми компонентами терминации трансс ляции у эукариот являются факторы eRF1 и eRF3 (см. обзор [1]). Белок eRF1 распознает любой из трех стоппкодонов (UAA, UAG или UGA) при его попадании в ААсайт рибосомы, что приводит к гидролизу связи пептидиллтРНК [2]. Фактор eRF3 – это GTPаза, которая стимулирует фактор eRF1, обеспечивая высвобождение новосинтезии рованной полипептидной цепи с рибосомы [3, 4]. Для терминации трансляции необходимо взаимоо * Эл. почта: zhouravleva@rambler.ru действие белков eRF1 и eRF3 [3, 5]. У дрожжей Saccharomyces сerevisiae фактор терминации трансс ляции eRF1 кодируется геном SUP45 [6, 7], а факк тор eRF3 – геном SUP35 [8–10]. Несмотря на то что гены SUP35 и SUP45 жизненно важны для клетки, показана возможность возникновения в них нонсенссмутаций [11–15]. Не будучи летальь ными, такие мутации приводят к нарушению терр минации трансляции. Впервые дрожжи с нонсенссмутацией в гене SUP45 получили на фоне доминантного супресс
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    ABSTRACT: The mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q --> TAA, 242R --> TGA, 317L --> TAG). The nonsense mutations in SUP45 gene were shown to be situated in moderate contexts for readthrough efficiency. We showed that readthrough efficiency of some of the mutations present in the sup45 mutants is not correlated with full-length Sup45 protein amount. This resulted from modification of both sup45 mRNA stability which varies 3-fold among sup45-n mutants and degradation rate of mutant Sup45 proteins. Our results demonstrate that some substitutions in the place of PTCs decrease Sup45 stability. The viability of sup45 nonsense mutants is therefore supported by diverse mechanisms that control the final amount of functional Sup45 in cells.
    MGG Molecular & General Genetics 04/2009; 282(1):83-96. DOI:10.1007/s00438-009-0447-5 · 2.83 Impact Factor
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    ABSTRACT: Proteins eRF1 and eRF3 are key components of translation termination in eukaryotes. The highly conserved translation termination factor eRF1 decodes stop codons, while another eukaryotic release factor (RF) eRF3 stimulates eRF1 in GTP-dependent manner. Functional C-terminal domain of eRF3 is necessary for cell viability and reveals high degree of similarity between all known eRF3 and elongation factor eEF1A. Unlike the C-terminal part, the N-terminal region of eRF3 proteins is not conserved and contains ‘prion forming domain’ (PFD). In mammals, eRF3 homologous proteins can be divided into two subfamilies based on the sequence of their N termini, GSPT1 (eRF3a) and GSPT2 (eRF3b). In our work we hypothesize that GSPT2 gene originated through retrotransposition of processed GSPT1 transcript after divergence between placental and marsupial mammals. Data obtained on the order Rodentia indicate that nucleotide sequence encoding N-terminal part of GSPT2 maybe used as a new marker for philogenetic analysis to distinguish between families.
    12/2007: pages 277-287;
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    ABSTRACT: Nonlethal nonsense mutations obtained earlier in the essential gene SUP45 encoding the translation termination eRFI factor in the yeast Saccharomyces cerevisiae were further characterized. Strains carrying these mutations retain the viability, since the full-length eRF1 protein is present in these strains, although in decreased amounts as compared to wild-type cells, together with a truncated eRF1. All nonsense mutations are likely to be located in a weak termination context, because a change in the stop codon UGAA (in the case of mutation sup45-107) to UAGA (sup45-107.2) led to the alteration of the local context from a weak to strong and to the lethality of the strain carrying sup45-107.2. All nonsense mutations studied are characterized by thermosensitivity expressed as cell mortality after cultivation at 37 degrees C. When grown under nonpermissive conditions (37 degrees C), cells of nonsense mutants sup45-104, sup45-105. and sup45-107 display a decrease in the amount of the truncated eRF1 protein without reduction in the amount of the full-length eRF1 protein. The results of this study suggest that the N-terminal eRF1 fragment is indispensable for cell viability of nonsense mutants due to the involvement in termination of translation.
    Genetika 11/2007; 43(10):1363-71. · 0.37 Impact Factor
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    ABSTRACT: Nonlethal nonsense mutations obtained earlier in the essential gene SUP45 encoding the translation termination factor eRF1 in the yeast Saccharomyces cerevisiae were further characterized. Strains carrying these mutations retain the viability, since the full-length eRF1 protein is present in these strains, although in decreased amounts as compared to wild-type cells, together with a trucated eRF1. All nonsense mutations are likely to be located in a weak termination context, because a change in the stop codon UGAA (in the case of mutation sup45-107) to UAGA (sup45-107.2) led to the alteration of the local context from a weak to strong and to the lethality of the strain carrying sup45-107.2. All nonsense mutations studied are characterized by thermosensitivity expressed as cell mortality after cultivation at 37°C. When grown under nonpermissive conditions (37°C), cells of nonsense mutants sup45-104, sup45-105, and sup45-107 display a decrease in the amount of the truncated eRF1 protein without reduction in the amount of the full-length eRF1 protein. The results of this study suggest that the N-terminal eRF1 fragment is indispensable for cell viability of nonsense mutants due to the involvement in termination of translation.
    Russian Journal of Genetics 10/2007; 43(10):1139-1146. DOI:10.1134/S1022795407100079 · 0.41 Impact Factor
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    ABSTRACT: The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3. In this work we have used previously isolated sup45 mutants of S. cerevisiae to characterize degradation of aberrant mRNA in conditions when translation termination is impaired. We have sequenced his7-1, lys9-A21 and trp1-289 alleles which are frequently used for analysis of nonsense suppression. We have established that sup45 nonsense and missense mutations lead to accumulation of his7-1 mRNA and CYH2 pre-mRNA. Remarkably, deletion of the UPF1 gene suppresses some sup45 phenotypes. In particular, sup45-n upf1Delta double mutants were less temperature sensitive, and more resistant to paromomycin than sup45 single mutants. In addition, deletion of either UPF2 or UPF3 restored viability of sup45-n double mutants. This is the first demonstration that sup45 mutations do not only change translation fidelity but also acts by causing a change in mRNA stability.
    BMC Molecular Biology 02/2007; 8:71. DOI:10.1186/1471-2199-8-71 · 2.06 Impact Factor
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    ABSTRACT: We have earlier characterized Saccharomyces cerevisiae strains with mutations of essential SUP45 and SUP35, which code for translation termination factors eRF1 and eRF3, respectively. In this work, the sup45 and sup35 nonsense mutants were compared with respect to the levels of eight tRNAs: tRNATyr, tRNAGln, tRNATrp, tRNALeu, tRNAArg (described as potential suppressor tRNAs), tRNAPro, tRNAHis, and tRNAGly. The mutants did not display a selective increase in tRNAs, capable of a noncanonical read-through at stop codons. Most of the mutations increased the level of all tRNAs under study. The mechanisms providing for the viability of the sup45 and sup35 nonsense mutants are discussed.
    Molecular Biology 06/2006; 40(4):647-653. DOI:10.1134/S0026893306040170 · 0.74 Impact Factor
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    ABSTRACT: Earlier we have characterized strains bearing mutations in essential genes SUP45 and SUP35 of yeast S. cerevisiae, encoding translation termination factors eRF1 and eRF3 respectively. In the present work nonsense-mutants on genes SUP45 and SUP35 have been compared by a level of eight tRNA: tRNATyr, tRNAGln, tRNATrp, tRNALeu and tRNAArg (previously described as potentially suppressor tRNA), and also tRNAPro, tRNAHis and tRNAGly. We have not revealed preferable increase in amount of natural suppressor tRNA. The majority of the investigated mutations leads to increase in a level of all investigated tRNA. The mechanisms providing viability of nonsense-mutants on essential genes SUP45 and SUP35 are discussed.
    Molekuliarnaia biologiia 01/2006; 40(4):724-30.
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    ABSTRACT: Collection of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1 has been obtained by different approaches. It has been shown that most of isolated mutations cause amino acid substitutions in the N-terminal part of eRF1 and do not decrease the eRF1 amount. Most of mutations studied do not abolish eRF1-eRF3 interaction. The role of the N-terminal part of eRF1 in stop codon recognition is discussed.
    Genetika 06/2004; 40(5):599-606. DOI:10.1023/B:RUGE.0000029148.58151.91 · 0.37 Impact Factor
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    ABSTRACT: Background Termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) – eRF1 and eRF3. The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45. Results We have isolated five sup45-n (n from nonsense) mutations that cause nonsense substitutions in the following amino acid positions of eRF1: Y53 → UAA, E266 → UAA, L283 → UAA, L317 → UGA, E385 → UAA. We found that full-length eRF1 protein is present in all mutants, although in decreased amounts. All mutations are situated in a weak termination context. All these sup45-n mutations are viable in different genetic backgrounds, however their viability increases after growth in the absence of wild-type allele. Any of sup45-n mutations result in temperature sensitivity (37°C). Most of the sup45-n mutations lead to decreased spore viability and spores bearing sup45-n mutations are characterized by limited budding after germination leading to formation of microcolonies of 4–20 cells. Conclusions Nonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors.
    BMC Molecular Biology 02/2003; 4. DOI:10.1186/1471-2199-4-2 · 2.06 Impact Factor
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    ABSTRACT: The termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs), eRF1 and eRF3. In mammals two genes encoding eRF3 structural homologues were identified and named GSPT1 and GSPT2. In the present study, we demonstrate that mouse mGSPT2 but not mGSPT1 could functionally substitute the essential yeast gene SUP35. However, we show that the complementation property of mGSPT1 protein is modified when NH2-tagged by GST. Since mGSPT1 and mGSPT2 differ mainly in their N-terminal regions, we developed a series of N-terminal deleted constructs and tested them for complementation in yeast. We found that at least amino acids spanning 84-120 of mGSPT1 prevent the complementation of sup35 mutation. The fact that chimeras between mGSPT1, mGSPT2 and yeast Sup35 complement the disruption of the SUP35 gene indicates that the N-terminal region of mGSPT1 is not sufficient by itself to prevent complementation. Complementation of the mutant with a double disruption of SUP35 and SUP45 genes is obtained when mGSPT2 and human eRF1 are co-expressed but not by co-expression of mGSPT1 and human eRF1. Our results strongly suggest that the two proteins (mGSPT1 and mGSPT2) are different. We hypothesize that the full length mGSPT1 does not have the properties expected for eRF3.
    Genes to Cells 11/2002; 7(10):1043-57. DOI:10.1046/j.1365-2443.2002.00585.x · 2.86 Impact Factor
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    ABSTRACT: Three 5S rRNA-binding ribosomal proteins (L5, L18, TL5) of extremely thermophilic bacterium Thermus thermophilushave earlier been isolated. Structural analysis of their complexes with rRNA requires identification of their binding sites in the 5S rRNA. Previously, a TL5-binding site has been identified, a TL5–RNA complex crystallized, and its structure determined to 2.3 . The sites for L5 and L18 were characterized, and two corresponding 5S rRNA fragments constructed. Of these, a 34-nt fragment specifically interacted with L5, and a 55-nt fragment interacted with L5, L18, and with both proteins. The 34-nt fragment–L5 complex was crystallized; the crystals are suitable for high-resolution X-ray analysis.
    Molecular Biology 06/2001; 35(4):521-526. DOI:10.1023/A:1010562707875 · 0.74 Impact Factor
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    ABSTRACT: Three 5S rRNA-binding ribosomal proteins (L5, L18, TL5) of extremely thermophilic bacterium Thermus thermophilus have earlier been isolated. Structural analysis of their complexes with rRNA requires identification of their binding sites in the 5S rRNA. Previously, a TL5-binding site has been identified, a TL5-RNA complex crystallized, and its structure determined to 2.3 A. The sites for L5 and L18 were characterized, and two corresponding 5S rRNA fragments constructed. Of these, a 34-nt fragment specifically interacted with L5, and a 55-nt fragment interacted with L5, L18, and with both proteins. The 34-nt fragment-L5 complex was crystallized; the crystals are suitable for high-resolution X-ray analysis.
    Molekuliarnaia biologiia 01/2001; 35(4):610-6.
  • Journal of Biomolecular NMR 08/2000; 17(3):273-4. DOI:10.1023/A:1008319622724 · 3.31 Impact Factor

Publication Stats

96 Citations
16.48 Total Impact Points

Institutions

  • 2004–2010
    • Saint Petersburg State University
      Sankt-Peterburg, St.-Petersburg, Russia
  • 2002–2003
    • Université de Rennes 1
      • Faculty of Medicine
      Roazhon, Brittany, France
  • 2000
    • Russian Academy of Sciences
      • Institute of Protein Research
      Moskva, Moscow, Russia